C3b generation is affected by the structure of the O-antigen polysaccharide in lipopolysaccharide from salmonellae

Salmonellae differing in the O-antigen side chain of their lipopolysaccharide were previously shown to activate the alternative pathway of complement to different extents. We now examine the generation of the major cleavage fragment of the complement component C3 (C3b) on these bacteria in a system that contains the purified components C3, B, D, and P but lacks the regulatory proteins H and I. The deposition of C3b in this system reproduces the same pattern obtained earlier with the use of whole serum, with the expected differences among the strains bearing different O-antigen. However, two distinct mechanisms for these differences in C3b generation became apparent. The intermediate activating strain showed 3 to 4 times less initial deposition of C3b than the other two strains. In contrast, the least activating strain showed adequate initial deposition but poor amplification, as shown by 2 to 3.4 lower amplification indexes as compared with those on the other two strains. Binding studies with factor B showed that decreased C3 convertase formation was responsible for the low amplification on this strain. Only 25% of the C3b bound to its surface was able to bind factor B with a high affinity, in comparison with 90% on the other two strains. No differences were found for the binding of factor H among the strains. These studies identify the molecular mechanisms by which these bacteria avoid complement activation.     

Mapping of the Locus for Escherichia coli Transfer Ribonucleic Acid Nucleotidyltransferase

cca Mutants of Escherichia coli K12 have altered levels of tRNA nucleotidyl-transferase activity. The cca locus has been located at minute 59.4 of the E. coli linkage map. It is cotransduced with tolC but not with argG, and is the earliest known marker transferred by Hfr strain KL14. The proximity of the tolC locus to the integrated sex factor in Hfr strain KL14 may be useful for mapping sex factor mutations by transduction.     

Inactivation of bacteriophages by protein E, a new major membrane protein isolated from an Escherichia coli mutant.

Pure protein E, obtained after diethylaminoethyl-cellulose chromatography of ethylenediaminetetraacetic acid-Triton X-100-solubilized outer membrane proteins of Escherichia coli strain JF694, inactivated bacteriophage K3. Lipopolysaccharide enhanced bacteriophage inactivation. Antibody prepared against purified protein E protected bacteriophage K3 from inactivation by protein E. Bacteriophage K3 used a major outer membrane protein, protein II*, as part of its receptor. We conclude that proteins E and II* have a common region which interacts with bacteriophage K3. Protein E also inactivated two recently described bacteriophages, TC45 and TC23, that use protein E as at least part of their receptor.     

IFN-gamma mediates the death of Th1 cells in a paracrine manner

Th1 cells have different capacities to develop into memory cells based on their production of IFN-gamma. In this study, the mechanism by which a homogenous population of IFN-gamma-producing CD4 T cells was eliminated in vivo was assessed. When such cells were transferred into naive mice and activated with Ag, a striking decrease in the frequency of cells in the spleen and lung was observed. However, administration of neutralizing anti-IFN-gamma Ab at the time of Ag challenge largely prevented the elimination of such cells. To determine whether IFN-gamma was mediating its effects directly and/or indirectly, the ability of IFN-gamma to effectively signal in such cells was assessed in vitro. Indeed, there was reduced phosphorylation of STAT1 in response to IFN-gamma as well as markedly reduced expression of the IFN-gammaR beta-chain. Furthermore, transfer of such cells into IFN-gammaR-deficient mice limited their death following activation with Ag. Together, these data suggest that IFN-gamma acts in a paracrine manner to mediate the death of activated IFN-gamma-producing Th1 cells. In contrast to Ag stimulation, administration of CpG alone resulted in the elimination of Th1 cells in IFN-gammaR-/- mice. These results show that in response to Ag stimulation, the death of IFN-gamma-producing effector Th1 cells is controlled in an IFN-gamma-dependent manner, whereas in response to innate activation, the death of IFN-gamma-producing Th1 cells can occur through an IFN-gamma-independent pathway. Collectively, these data show the multiple mechanisms by which Th1 effector cells are efficiently eliminated in vivo.     

Leaf mechanical properties modulate feeding movements and ingestive success of the pond snail, Lymnaea stagnalis

We examined the mechanical properties of Butterhead and Iceberg lettuce leaves, and the rate at which they were eaten by the pond snail Lymnaea stagnalis. The outer part of Butterhead leaves were less robust than either the inner Butterhead or outer Iceberg leaves (Young’s modulus 2.8, 5.2, 7.7 MPa respectively; ultimate tensile stress 0.18, 0.34 0.51 MPa) which were also thicker. Snails ingested inner Butterhead and Iceberg strips more slowly (36 and 32%) than outer Butterhead. This was not due to differences in latency to first bite or biting rate. Rather, the drop was due to a decrease in the proportion of successful bites (inner Butterhead 84%; Iceberg 86%), to a shorter length ingested per bite (inner Butterhead 55%; Iceberg 45%) and to increased handling time (inner Butterhead 30%). We conclude that sensory input from the mechanically more robust lettuce slows the buccal central pattern generator.     

Synthesis, characterization, and evaluation of ferrocene-theophylline conjugates for use in electrochemical enzyme immunoassay

A series of 8-(ferrocenylalkyl)theophylline conjugates were synthesized for evaluation in a homogeneous, competitive electrochemical immunoassay for theophylline with amperometric detection of the ferrocene label at +320 mV. The electrical signal was amplified via redox cycling with the glucose oxidase/glucose system. The resulting catalytic current was strongly inhibited upon binding of the conjugates to anti-theophylline antibodies such that a large excess of theophylline was required to achieve complete reversal leading to an assay with poor sensitivity in the clinical range. A study of the nonspecific interaction of the antibodies with various ferrocene derivatives indicated that this was reduced when a charged functional group was present on the metallocene ring. Consequently, a conjugate was synthesized with a quaternary ammonium group which when incorporated into the assay resulted in improved sensitivity.     

Identification of phylogenetic trees of minimal length.

The problem of determining an optimal phylogenetic tree from a set of data is an example of the Steiner problem in graphs. There is no efficient algorithm for solving this problem with reasonably large data sets. In the present paper an approach is described that proves in some cases that a given tree is optimal without testing all possible trees. The method first uses a previously described heuristic algorithm to find a tree of relatively small total length. The second part of the method independently analyses subsets of sites to determine a lower bound on the length of any tree. We simultaneously attempt to reduce the total length of the tree and increase the lower bound. When these are equal it is not possible to make a shorter tree with a given data set and given criterion. An example is given where the only two possible minimal trees are found for twelve different mammalian cytochrome c sequences. The criterion of finding the smallest number of minimum base changes was used. However, there is no general method of guaranteeing that a solution will be found in all cases and in particular better methods of improving the estimate of the lower bound need to be developed.